RASGRP1 targeted by H3K27me3 regulates myoblast proliferation and differentiation in mice and pigs

Skeletal muscle is not only the largest organ in the body that is responsible for locomotion and exercise but also crucial for maintaining the body’s energy metabolism and endocrine secretion. The trimethylation of histone H3 lysine 27 (H3K27me3) is one of the most important histone modifications that participates in muscle development regulation by repressing the transcription of genes. Previous studies indicate that the RASGRP1 gene is regulated by H3K27me3 in embryonic muscle development in pigs, but its function and regulatory role in myogenesis are still unclear. In this study, we verify the crucial role of H3K27me3 in RASGRP1 regulation. The gain/loss function of RASGRP1 in myogenesis regulation is performed using mouse myoblast C2C12 cells and primarily isolated porcine skeletal muscle satellite cells (PSCs). The results of qPCR, western blot analysis, EdU staining, CCK-8 assay and immunofluorescence staining show that overexpression of RASGRP1 promotes cell proliferation and differentiation in both skeletal muscle cell models, while knockdown of RASGRP1 leads to the opposite results. These findings indicate that RASGRP1 plays an important regulatory role in myogenesis in both mice and pigs.


Introduction
Skeletal muscle is not only the largest organ responsible for animal locomotion and exercise capacity but also crucial for maintaining the body's energy metabolism and endocrine secretion [1][2][3].In livestock-producing fields, skeletal muscle is of particular importance, as meat is the main animal product, in addition to animal fur, milk and others.Increasing evidence indicates that epigenetic regulators participate in the development of skeletal muscle.
In this study, we first confirmed that the expression of the RASGRP1 gene was regulated by H3K27me3 enrichment in its promoter during skeletal muscle development.Then, we studied the function of RASGRP1 during cell proliferation and differentiation in both mouse C2C12 myoblasts and PSCs.Our results demonstrated that RASGRP1 is regulated by H3K27me3 and promotes myogenesis in pigs and mice.

Materials and Methods Animals
Pigs, including Duroc sows for embryonic skeletal muscle collection and newborn piglets for isolating porcine skeletal muscle satellite cells, were purchased from Guangdong Wen's Foodstuffs Group (Yunfu, China).All animal experiments were conducted following the requirements for the Care and Use of Laboratory Animals by the Ethics Committee of the Laboratory Animal Center of South China Agricultural University, Guangzhou, China (Permit Number 2021F036, Permit Date 2 March 2021) [37].

Cell culture
C2C12 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).Dulbecco's modified Eagle's medium (DMEM) high glucose (Gibco, Grand Island, USA) and 10% fetal bovine serum (FBS; Gibco) was used for cell culture.For cell differentiation, DMEM high glucose with 2% horse serum (Gibco) was used.The cells were cultured with 5% CO 2 at 37°C.

Isolation and culture of porcine skeletal muscle satellite cells
Porcine skeletal muscle satellite cells (PSCs) were isolated from oneweek-old piglets and cultivated according to protocols described in previous studies [38,39].The tissues were digested with 2 mg/mL type II collagenase (Sigma-Aldrich, Darmstadt, Germany) and then placed in a 37°C incubator for 2.5 h.Then, the digestion was stopped with an equal volume of RPMI 1640 medium (Gibco) containing 1% penicillin-streptomycin (P-S; Gibco).Then, 100, 200, and 400-mesh sieves were used to filter the cell suspension.Cell proliferation medium was prepared according to a previous study and used to resuspend cells [39].Differential adhesion was used to obtain purified cells after 2 h of culture.When cells reached 70%-80% confluence, differentiation medium consisting of DMEM high glucose with 2% horse serum and 1% P-S was used to induce cell differentiation.

Quantitative real-time polymerase chain reaction (qPCR)
Total RNA kit ll (Omega Biotek, Norcross, USA) was used to harvest the total RNA following the manufacturer's protocol.cDNA was prepared using a PrimeScript RT reagent kit (TaKaRa, Tokyo, Japan) and used to perform qPCR in a Quant Studio 7 Flex system (Thermo Fisher, Scientific, Waltham, USA).All primers used are presented in Supplementary Table S1.

Chromatin immunoprecipitation (ChIP)
ChIP was conducted according to the protocol in a previous study [40].Briefly, micrococcal nuclease was used to fragment chromatin.After that, the chromatin fragments were incubated with magnetic beads (Biogle, Wuxi, China).The DNA-bead complex was immunoprecipitated with anti-H3K27me3 antibody (Millipore, Billerica, USA) or negative control IgG.The primers for the promoter of the RASGRP1 gene used for ChIP-qPCR were as follows: forward primer 5′-CTCTCCGAATTCCCCATTGTG-3′ and reverse primer 5′-AAATCAGAGCTGCATCCAC-3′.

Cell proliferation and cell cycle assays
For flow cytometry analysis, C2C12 cells stored at -20°C were fixed in 70% (v/v) ethanol overnight and treated at 4°C in 50 mg/mL propidium iodide for 30 min.Cell cycle detection was conducted in accordance with a previous study [39] by using a flow cytometer (Becton Dickinson, Franklin Lakes, USA).The cells were transfected with RASGRP1 siRNA or overexpression vectors when they reached approximately 50% confluence.CCK-8 assays (Yeasen, Shanghai, China) were conducted following the manufacturer's protocol after transfection.EdU staining was performed following the manufacturer's protocol (RiboBio, Guangzhou, China).Cells were to fixed and permeabilized separately with 4% paraformaldehyde and 0.5% Triton X-100, respectively.DAPI was used to stain

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RASGRP1 regulates myoblast proliferation and differentiation cell nuclei.All images were captured with the TE2000U fluorescence microscope imaging system (Nikon, Tokyo, Japan).

Immunofluorescence staining
Cells seeded in a 24-well plate were transfected with RASGRP1 siRNA or overexpression vectors when they reached approximately 50%-70% confluence.Then, 4% paraformaldehyde and 0.5% Triton X-100 were separately used to fix and permeabilize the cells for 30 min three days after transfection.Then, the cells were blocked in QuickBlock Buffer (Beyotime) at 37°C for 2 h and treated with the anti-MyHC antibody (sc-376157; 1:200; Santa Cruz Biotechnology) at 4°C overnight.Then, the cells were incubated with Alexa Fluor 488 fluorescent antibody (1:500; Thermo Fisher Scientific) after wash with PBS (Servicebio).DAPI was used to stain cell nuclei.All images were captured with the TE2000U fluorescence microscope imaging system.

Statistical analysis
Data are presented as the mean±standard error of the mean (SEM).
The comparative threshold cycle (2 -ΔΔct ) method was used to quantify the relative mRNA levels [38,41].Statistical analysis between different groups was performed by using two-tailed Student's t-test or one-way analysis of variance (ANOVA) in SPSS software (version 22.0).ImageJ software was used for the quantification of positively-stained cells and the visualization of western blots.All experiments were performed in triplicate.P<0.05 indicated significant difference.

RASGRP1 is targeted by H3k27me3
We collected longissimus dorsi muscles on gestation days 33, 65, and 90, which represent three critical developmental time points for embryonic myogenesis [42][43][44][45].ChIP-qPCR assay in skeletal muscles at E33, E65, and E90 showed that the enrichment of H3K27me3 in the RASGRP1 promoter was highest at E33 and lowest at E90 (Figure 1A).The mRNA expression of the RASGRP1 gene significantly increased during embryonic development (Figure 1B and Supplementary Figure S1), while its expression was pretty low in adult skeletal muscle.These results indicated that during embryonic porcine myogenesis, increased RASGRP1 expression might be specifically regulated by H3K27me3.

RASGRP1 promotes the proliferation of C2C12 cells
To investigate the role of the RASGRP1 gene in C2C12 cell proliferation, we performed gain-and loss-of-function analyses of the RASGRP1 gene.We overexpressed the RASGRP1 gene in C2C12 cells, and the qPCR results demonstrated that the relative expression of the RASGRP1 gene and the proliferation marker genes CDK2 and CDK14 were significantly increased (Figure 2A).CCK-8 assay demonstrated that RASGRP1 overexpression also significantly promoted the proliferation of C2C12 cells (Figure 2B).The relative protein expressions of the proliferation marker genes Ki67 and CDK2 were also increased, according to western blot analysis (Figure 2C).EdU staining revealed that RASGRP1 overexpression dramatically increased the percentage of EdU-positive (EdU + ) cells (Figure 2D).In addition, flow cytometry analysis showed that there were 5.33% more S-phase cells among the cells overexpressing RASGRP1 than in the pcDNA3.1(+)control group (Figure 2E).All the above results indicated that RASGRP1 promoted C2C12 cell proliferation.
To verify the effect of RASGRP1 overexpression on C2C12 cell proliferation, we then knocked down RASGRP1 by siRNA in C2C12 cells and detected its effect on C2C12 cell proliferation.The qPCR results showed that the relative expression of RASGRP1 as well as CDK2 and CDK14 was significantly reduced after RASGRP1 knockdown (Figure 3A).CCK-8 assay demonstrated that RASGRP1 knockdown also dramatically reduced the proliferation activity of C2C12 cells (Figure 3B).The relative protein expression of the Ki67 gene was markedly decreased (Figure 3C).EdU staining revealed that RASGRP1 knockdown dramatically reduced the percentage of EdU + cells (Figure 3D).These results indicated that RASGRP1 knockdown inhibited the proliferation of C2C12 cells, which was opposite to the positive effect of RASGRP1 overexpression on the proliferation of C2C12 cells.

RASGRP1 promotes the differentiation of C2C12 cells
To explore the function of RASGRP1 in C2C12 cell differentiation, we examined the expression profiles of RASGRP1 as well as the MyHC and MyoG genes during myogenic differentiation.The qPCR results demonstrated that the expression of the RASGRP1 gene was significantly upregulated during the differentiation of C2C12 cells, while the expressions of the MyHC gene and MyoG gene were also upregulated significantly (Figure 4A,B), indicating that RASGRP1 regulated the differentiation of C2C12 cells.Then, we overexpressed RASGRP1 to explore its regulatory effect on C2C12 cell differentiation.qPCR results demonstrated that the expression of RASGRP1 and differentiation marker genes (MyoG, MyoD and MyHC) was significantly increased after RASGRP1 overexpression (Figure 4C).The relative protein expressions of MyoG and MyHC were markedly increased after RASGRP1 overexpression (Figure 4D).The MyHC immunofluorescence assay results showed that 22.23% more fusion myotubes were present in the cells overexpressing RASGRP1 than in the control group (Figure 4E).These results suggested that RASGRP1 enhanced the differentiation of C2C12 cells.
To further verify the effect of RASGRP1 in regulating skeletal cell differentiation, we performed RASGRP1 knockdown experiments by siRNA in C2C12 cells.After transfection with siRNA for three days, total RNA and protein were extracted.The qPCR results demonstrated that the relative expressions of the RASGRP1 and MyHC genes were significantly decreased after siRNA transfection

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RASGRP1 regulates myoblast proliferation and differentiation (Figure 5A).The relative protein expressions of the MyoD and MyoG genes were also decreased after RASGRP1 knockdown (Figure 5B).The MyHC immunofluorescence assay results showed that there were 8.14% fewer fusion myotubes in the cells with RASGRP1 gene knockdown than in the control group (Figure 5C).All these results indicated that RASGRP1 knockdown inhibited C2C12 cell differentiation.

RASGRP1 promoted the proliferation and differentiation of PSCs
To investigate the role of RASGRP1 in PSC proliferation and differentiation, we also studied the function of RASGRP1 in pigs.After RASGRP1 overexpression, the qPCR results demonstrated that the relative expressions of the RASGRP1 gene and proliferation marker genes were significantly increased in PSCs (Figure 6A).CCK-8 assay demonstrated that RASGRP1 overexpression also significantly promoted the proliferation of PSCs (Figure 6B).EdU staining showed that RASGRP1 overexpression dramatically increased the percentage of EdU + cells (Figure 6C).Knockdown of RASGRP1 led to a significant decrease in the expressions of RASGRP1 gene and proliferation marker genes (Figure 6D).CCK-8 assay demonstrated that RASGRP1 knockdown significantly reduced the proliferative activity of PSCs (Figure 6E).EdU staining showed that RASGRP1 knockdown significantly reduced the percentage of EDU + cells (Figure 6F).We also detected the role of RASGRP1 in PSC differentiation.In PSCs, the expression level of the RASGRP1 gene was significantly increased during differentiation (Figure 6G).MyHC immunofluorescence assay results revealed that the fusion rate of myotubes was markedly increased/decreased after RASGRP1 overexpression/knockdown (Figure 6H,I).These findings suggested that RASGRP1 stimulated the proliferation and differentiation of PSCs.

Discussion
The RASGRP1 gene is one of the Ras gene families whose proteins can coordinate responses within each cell by detecting signals from other parts of the body [46][47][48] and self-regulate once their activity is altered, which may lead to the occurrence of cancer and RASGRP1 regulates myoblast proliferation and differentiation 5 developmental diseases [49].Increasing numbers of studies have revealed a connection between the RASGRP1 gene and muscle growth.RASGRP1 can affect the body size trait of pigs and exhibits a strong positive correlation with the body height and tube circumference traits of Suhuai pigs [50].In addition, deletion of the RASGRP1 gene inhibits the activation of ERK, which is a part of the classic MAPK/ERK pathway for regulating skeletal muscle proliferation [51].Furthermore, RASGRP1 was reported to be related to the regulation of p-ERK in the PPAR β/δ signaling pathway [52], which is widely involved in regulating myogenic and adipogenic differentiation [53].These results indicated that RASGRP1 may be involved in muscle development.However, the detailed function of RASGRP1 in myogenesis is largely unknown.H3K27me3 modification is widely distributed throughout the genome of myoblasts or myotubes and regulates muscle development as a silent marker mainly located at the promoter of genes [54,55].Previous studies also demonstrated that myogenic transcription factors were repressed by H3K27me3 during cell prolifera-

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RASGRP1 regulates myoblast proliferation and differentiation tion in PSCs, while H3K27me3 depletion promoted myogenic differentiation [55].Our previous sequencing results [36] revealed that RASGRP1 has a significant H3K27me3 enrichment peak in its promoter.Thus, we aimed to investigate the function of RASGRP1 in myogenesis and explore the regulatory function of H3K27me3 in RASGRP1.In this study, we confirmed that the enrichment level of H3K27me3 on the RASGRP1 gene decreased in pig embryonic skeletal muscle at E33, E65 and E90, which showed a significant negative correlation with RASGRP1 gene expression during pig embryonic muscle development.
We then examined the functions of the RASGRP1 gene in myogenesis.Since cell maturation is based on myoblast proliferation and myoblast differentiation is essential for muscle development and maturity, we examined the expression changes of some proliferation markers after loss/gain of RASGRP1 both in C2C12 cells and PSCs, such as Ki67, cyclin-dependent kinases (CDKs) and proliferating cell nuclear antigen (PCNA).Ki67 is often used as a key indicator and marker gene for cell proliferation in clinical studies [56], and CDKs participate in the regulation of the cell proliferation cycle [57].PCNA plays an important role in cell replication and promotes cell proliferation.Our results showed that RASGRP1 significantly promoted the relative expression of the above proliferation marker genes, which indicated its positive effect on cell proliferation.As expected, consistent results were obtained in cell proliferation assays.We found that the expression of the RASGRP1 gene increased continuously in the process of myoblast differentiation, as did that of myocyte differentiation marker genes.After overexpression of RASGRP1, the relative expressions of differentiation marker factors were also upregulated significantly.
The results of the MyHC immunofluorescence experiment showed that the number of differentiated myotube fusions was increased, indicating the promotion of cell differentiation.Consistently, the opposite trend was observed after RASGRP1 knockdown.These results suggested that RASGRP1 regulates skeletal muscle development by affecting the expressions of proliferation and differentiation factors, and this function may be conserved in mice and pigs.
In conclusion, we found that the promoter of the RASGRP1 gene is enriched by H3K27me3.The loss of H3K27me3 enrichment in RASGRP1 can promote its transcriptional activity during the development of skeletal muscle.Meanwhile, up/downregulation of RASGRP1 expression can promote/repress the proliferation and differentiation of PSCs and C2C12 cells.These results suggest that RASGRP1 has a positive regulatory role in myogenesis.

Figure 3 .
Figure 3. RASGRP1 knockdown inhibits the proliferation of C2C12 cells (A) The relative expression levels of the RASGRP1 gene and proliferation marker genes after RASGRP1 knockdown.(B) Detection of C2C12 cell viability by CCK-8 assay at 18 h and 24 h after RASGRP1 knockdown.(C) The relative protein expression levels of the Ki67 and CDK2 genes after RASGRP1 knockdown.(D) EdU staining after RASGRP1 knockdown in C2C12 cells.Scale bar: 100 μm.*P<0.05,**P<0.01.

Figure 4 .
Figure 4. RASGRP1 overexpression promotes the differentiation of C2C12 cells (A) Expression levels of differentiation marker genes during differentiation.(B) The relative expression level of RASGRP1 was increased significantly and then decreased during differentiation.(C) The relative expression levels of RASGRP1 and differentiation marker genes after RASGRP1 overexpression.(D) The relative protein expression of MyHC, MyoD and MyoG after RASGRP1 overexpression.(E) MyHC immunofluorescence assay after RASGRP1 overexpression in C2C12 cells.Scale bar: 50 μm.*P<0.05,**P<0.01.

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RASGRP1 regulates myoblast proliferation and differentiation Xiao et al.Acta Biochim Biophys Sin 2024